Treatment of congestive heart failure

ABSTRACT

A mammal with congestive heart failure is treated by administering to the mammal an effective amount of growth hormone. Treatment results in increased left ventricular cystolic pressure, increased left ventricular maximum, increased cardiac output, and increased stroke volume index. Treatment also results in reduced left ventricular end-diastolic pressure and reduced systemic vascular resistance. These measurements indicate improvement in cardiac function by increased ventricular contractility and decreased peripheral vascular resistance.

RELATED APPLICATIONS

This is a non-provisional continuation application of co-pendingapplication(s) Ser. No. 10/848,814 filed on May 18, 2004, which is acontinuation of Ser. No. 10/623,129 filed on Jul. 18, 2003, which is acontinuation of Ser. No. 10/288,254 filed Nov. 4, 2002, which is acontinuation of Ser. No. 10/045,622 filed on Oct. 24, 2001, which is acontinuation of Ser. No. 09/724,787 filed on Nov. 28, 2000, which is acontinuation of Ser. No. 09/550,736, filed Apr. 17, 2000, which is acontinuation of Ser. No. 09/302,924, filed Apr. 30, 1999, which is acontinuation of application Ser. No. 08/228,548, filed Apr. 15, 1994,now U.S. Pat. No. 5,935,924 which application(s) is(are) incorporatedherein by reference and to which application(s) priority is claimedunder 35 USC § 120.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of treating patients havingcongestive heart failure with growth hormone (GH).

2. Description of Background and Related Art

In vitro studies have shown that chronic hypersecretion of growthhormone by implantation of a growth hormone-secreting tumor isassociated with an increase in the maximum isometric force of leftventricular papillary muscle normalized per cross-sectional area and nochange in both the unloaded shortening velocity of the isolated muscleand the calcium and actin-activated myosin in normal rats. This isobserved despite a marked shift of the isomyosin pattern toward the lowATPase activity V3 isoform. These results suggest that growth hormonemay induce a unique pattern of myocardial contraction: a normalshortening speed and an increased force generation are associated withchanges in myosin phenotype that allow the cardiac muscle to functionmore economically. Timsit, J. et al., J. Clin. Invest. 86: 507-515(1990); Timsit, J. et al., Acta Paediatr Suppl 383: 32-34 (1992).

In vitro studies from the same investigators were carried out on ratcardiac skinned fibers. These studies have demonstrated that thecontractile performance of the skinned fiber from rat myocardiumsubjected to chronically high circulating growth hormone levels isincreased. The increase in contractile performance was shown to be dueto specific alterations in the properties of the contractile apparatus,including an increase in both maximal tension and myofibrillarsensitivity to calcium. Mayoux, E. et al., Circulation Research 72(1):57-64 (1993).

It has been found that left ventricular dP/dt, cardiac index, strokeindex, and stroke work are significantly increased inchloralose-urethan-anesthetized rats with a transplantable growth growthhormone-secreting tumor. The data suggest that chronic hypersecretion ofgrowth hormone increases cardiac output by increasing contractility inanesthetized rats. Penney, D. G. et al., Cardiovascular Research 19:270-277 (1985).

A contrary result has also been reported. Rubin, S. A. et. al., J. Mol.Cell Cardiol. 22: 429-438 (1990) have reported that similar chronichypersecretion of growth hormone induced by the growth hormone-secretingtumor causes significant decreases in left ventricular contractility(maximum dP/dt) and increases in LVEDP in ketamine-anesthetized rats.

In a clinical study, it has been shown that administration of humangrowth hormone to normal subjects for one week increases ventricularcontractility and cardiac output, as evaluated by echocardiography.Thuesen, L et. al., Dan. Med. Bull. 35: 193-196 (1988).

In adults with growth hormone deficiency, growth hormone treatmentproduced significant increases in stroke volume and exercise capacity.These results suggest that growth hormone can improve cardiac functionat rest and during exercise in the adult patients with growth hormonedeficiency. Jorgensen, J. et. al., Lancet i: 1221-1225 (1989); Cuneo, R.et. al., J. Appl. Physiol. 70: 695-700 (1991); Christiansen, J. S. et.al., Acta Paediatr Suppl 383: 40-42 (1992).

Cuneo et. al., Lancet i: 838-839 (1989) have reported a very interestingcase showing effects of growth hormone therapy in a patient withextremely poor cardiac function. The patient developed severe heartfailure eight months after hypophysectomy for Cushing syndrome. Standardtherapy including diuretics and angiotensin-converting enzyme inhibitorshad little effect and cardiac transplantation was considered. As a lastresort, treatment with human growth hormone, 12 IU/day s.c. was tried,with a remarkable beneficial effect. Clinical improvement and increasesin myocardial contractility and cardiac output were noted.

Until now, however, effects of human growth hormone in heart failurepatients without growth hormone deficiency have not been reported, toapplicants' knowledge. Heart failure affects approximately three millionAmericans, developing in about 400,000 each year. Current therapy forheart failure is insufficient. Although angiotensin converting enzyme(ACE) inhibitors have been shown to have beneficial effects in patientswith heart failure, they appear consistently unable to relieve symptomsin more than 60% of heart failure patients. In addition, they reducemortality of heart failure only by approximately 15-20%. Therefore,there is room for improvement in the therapy of heart failure.

Accordingly it is an object of this invention to provide a method oftreatment for a patient with congestive heart failure.

SUMMARY

The present invention achieves this object by the provision of a methodof treatment of congestive heart failure, the method characterized byadministration of an effective amount of growth hormone (GH). Theadministration of GH results in improvement of cardiac function byincreased ventricular contractility and decreased peripheral vascularresistance.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a. Shows the body weight before treatment in ligated and shamcontrols. **P<0.01, compared to the respective vehicle group.

FIG. 1 b. Shows an increase in body weight following treatment inligated and sham controls. **P<0.01, compared to the respective vehiclegroup.

FIG. 1 c. Shows a comparison of the increase in the ratio of ventricularweight to body weight in ligated and sham controls. **P<0.01, comparedto the respective vehicle group.

FIG. 2 a. Shows the effect of GH administration on serum levels of GH inligated and sham controls. **P<0.01, compared to the respective vehiclegroup.

FIG. 2 b. Shows the effect of GH administration on serum levels of IGF-1in ligated and sham controls. **P<0.01, compared to the respectivevehicle group.

FIG. 3 a. Shows the effects of GH and vehicle on mean arterial pressure(MAP) in ligated rats and sham controls, #P<0.05, ## P<0.01, compared tothe respective sham group. *P<0.05, compared to the respective vehiclegroup.

FIG. 3 b. Shows the effects of GH on systolic arterial pressure (SAP) inligated rats and sham controls. #P<0.05, ## P<0.01, compared to therespective sham group. *P<0.05, compared to the respective vehiclegroup.

FIG. 3 c. Shows the effects of GH on heart rate (HR) in ligated rats andsham controls. #P<0.05, ## P<0.01, compared to the respective shamgroup. *P<0.05, compared to the respective vehicle group.

FIG. 4 a. Shows the effects of GH on left ventricular maximum dP/dt.*P<0.05, **P<0.01, compared to the respective vehicle group. # P<0.05,## P<0.01, compared to the respective sham group.

FIG. 4 b. Shows the effects of growth hormone (GH) on left ventricularsystolic pressure (LVSP). *P<0.05, **P<0.01, compared to the respectivevehicle group. # P<0.05, ## P<0.01, compared to the respective shamgroup.

FIG. 4 c. Shows the effects of growth hormone (GH) on left ventricularend-diastolic pressure (LVEDP). *P<0.05, **P<0.01, compared to therespective vehicle group. # P<0.05, ## P<0.01, compared to therespective sham group.

FIG. 5 a. Shows the effects of growth hormone (GH) on cardiac index (CI)in ligated rats and sham controls. *P<0.05, compared to the respectivevehicle group. #P<0.05, compared to the respective sham group.

FIG. 5 b. Shows the effects of growth hormone (GH) on stroke volumeindex, (SVI) in ligated rats and sham controls. *P<0.05, compared to therespective vehicle group. #P<0.05, compared to the respective shamgroup.

FIG. 5 c. Shows the effects of growth hormone (GH) on systemic vascularresistance (SVR) in ligated rats and sham controls. *P<0.05, compared tothe respective vehicle group. #P<0.05, compared to the respective shamgroup.

DETAILED DESCRIPTION OF THE INVENTION

a. Definitions

In general, the following words or phrases or abbreviations have theindicated definition when used in the description, examples, and claims:

As used herein, “BW” refers to body weight.

As used herein, “CO” refers to cardiac output.

As used herein, “CI” refers to cardiac index. The cardiac index ismeasurable as cardiac output divided by body weight (CO/BW).

As used herein, “dP/dt” refers to left ventricular maximum. As usedherein, “HR” refers to heart rate.

As used herein, “LCA” refers to left coronary artery.

As used herein, “LVEDP” refers to left ventricular end-diastolicpressure.

As used herein, “LVMP” refers to left ventricular mean pressure.

As used herein, “LVSP” refers to left ventricular systolic pressure. Asused herein, “MAP” refers to mean arterial pressure.

As used herein, “RAP” refers to right atrial pressure.

As used herein, “SAP” refers to systolic arteriol pressure.

As used herein, “SV” refers to stroke volume. The stroke volume ismeasurable as CO/HR.

As used herein, “SVI” refers to stroke volume index. The stroke volumeindex is measurable as SV/BW.

As used herein, “SVR” refers to systemic vascular resistance. The SVR ismeasurable as MAP/CI.

As used herein, “VW” refers to ventricular weight.

As used herein “infarct” refers to an area of necrosis resulting from aninsufficiency of blood supply. “Myocardial infarction” refers tomyocardial necrosis resulting from the insufficiency of coronary bloodsupply.

As used herein “treatment” refers to ameliorating the congestive heartfailure condition.

As used herein “congestive heart failure” refers to

As used herein, the term “mammal” refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal herein is human.

As used herein, “growth hormone” or “GH” refers to growth hormone innative-sequence or in variant form, and from any source, whethernatural, synthetic, or recombinant. Examples include human growthhormone (hGH), which is natural or recombinant GH with the human nativesequence (somatotropin or somatropin), and recombinant growth hormone(rGH), which refers to any GH or variant produced by means ofrecombinant DNA technology, including somatrem, somatotropin, andsomatropin. Preferred herein for human use is recombinant humannative-sequence, mature GH with or without a methionine at itsN-terminus. More preferred is methionyl human growth hormone (met-hGH)produced in E. coli, e.g., by the process described in U.S. Pat. No.4,755,465 issued Jul. 5, 1988 and Goeddel et al., Nature, 282: 544(1979). Met-hGH, which is sold under the trademark Protropin® byGenentech, Inc., is identical to the natural polypeptide, with theexception of the presence of an N-terminal methionine residue. Thisadded amino acid is a result of the bacterial protein synthesis process.Also preferred is recombinant hGH available from Genentech, Inc. underthe trademark Nutropin®. This latter hGH lacks this methionine residueand has an amino acid sequence identical to that of the natural hormone.See Gray et al., Biotechnology, 2: 161 (1984). Both methionyl hGH andhGH have equivalent potencies and pharmacokinetic values. Moore et al.,Endocrinology, 122: 2920-2926 (1988). Another appropriate hGH candidateis an hGH variant that is a placental form of GH with pure somatogenicand no lactogenic activity as described in U.S. Pat. No. 4,670,393issued 2 Jun. 1987. Also included are GH variants as described in WO90/04788 published 3 May 1990 and WO 92/09690 published 11 Jun. 1992.

b. Modes for Carrying Out the Invention

Therapeutic Compositions and Administration of GH

Therapeutic formulations of GH are prepared for storage by mixing GHhaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, supra), in the form of lyophilized cake oraqueous solutions. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

For administration, GH may be complexed or bound to a polymer toincrease its circulatory half-life. Examples of polyethylene polyols andpolyoxyethylene polyols useful for this purpose include polyoxyethyleneglycerol, polyethylene glycol, polyoxyethylene sorbitol, polyoxyethyleneglucose, or the like. The glycerol backbone of polyoxyethylene glycerolis the same backbone occurring in, for example, animals and humans inmono-, di-, and triglycerides.

The polymer need not have any particular molecular weight, but it ispreferred that the molecular weight be between about 3500 and 100,000,more preferably between 5000 and 40,000. Preferably the PEG homopolymeris unsubstituted, but it may also be substituted at one end with analkyl group. Preferably, the alkyl group is a C₁-C₄ alkyl group, andmost preferably a methyl group. Most preferably, the polymer is anunsubstituted homopolymer of PEG, a monomethyl-substituted homopolymerof PEG (mPEG), or polyoxyethylene glycerol (POG) and has a molecularweight of about 5000 to 40,000.

The GH is covalently bonded via one or more of the amino acid residuesof the GH to a terminal reactive group on the polymer, depending mainlyon the reaction conditions, the molecular weight of the polymer, etc.The polymer with the reactive group(s) is designated herein as activatedpolymer. The reactive group selectively reacts with free amino or otherreactive groups on the GH. It will be understood, however, that the typeand amount of the reactive group chosen, as well as the type of polymeremployed, to obtain optimum results, will depend on the particular GHemployed to avoid having the reactive group react with too manyparticularly active groups on the GH. As this may not be possible toavoid completely, it is recommended that generally from about 0.1 to1000 moles, preferably 2 to 200 moles, of activated polymer per mole ofprotein, depending on protein concentration, is employed. The finalamount of activated polymer per mole of protein is a balance to maintainoptimum activity, while at the same time optimizing, if possible, thecirculatory half-life of the protein.

While the residues may be any reactive amino acids on the protein, suchas one or two cysteines or the N-terminal amino acid group, preferablythe reactive amino acid is lysine, which is linked to the reactive groupof the activated polymer through its free epsilon-amino group, orglutamic or aspartic acid, which is linked to the polymer through anamide bond.

The covalent modification reaction may take place by any appropriatemethod generally used for reacting biologically active materials withinert polymers, preferably at about pH 5-9, more preferably 7-9 if thereactive groups on the GH are lysine groups. Generally, the processinvolves preparing an activated polymer (with at least one terminalhydroxyl group), preparing an active substrate from this polymer, andthereafter reacting the GH with the active substrate to produce the GHsuitable for formulation. The above modification reaction can beperformed by several methods, which may involve one or more steps.Examples of modifying agents that can be used to produce the activatedpolymer in a one-step reaction include cyanuric acid chloride(2,4,6-trichloro-S-triazine) and cyanuric acid fluoride.

In one embodiment the modification reaction takes place in two stepswherein the polymer is reacted first with an acid anhydride such assuccinic or glutaric anhydride to form a carboxylic acid, and thecarboxylic acid is then reacted with a compound capable of reacting withthe carboxylic acid to form an activated polymer with a reactive estergroup that is capable of reacting with the GH. Examples of suchcompounds include N-hydroxysuccinimide, 4-hydroxy-3-nitrobenzenesulfonic acid, and the like, and preferably N-hydroxysuccinimide or4-hydroxy-3-nitrobenzene sulfonic acid is used. For example, monomethylsubstituted PEG may be reacted at elevated temperatures, preferablyabout 100-110° C. for four hours, with glutaric anhydride. Themonomethyl PEG-glutaric acid thus produced is then reacted withN-hydroxysuccinimide in the presence of a carbodiimide reagent such asdicyclohexyl or isopropyl carbodiimide to produce the activated polymer,methoxypolyethylene glycolyl-N-succinimidyl glutarate, which can then bereacted with the GH. This method is described in detail in Abuchowski etal., Cancer Biochem. Biophys., 7: 175-186 (1984). In another example,the monomethyl substituted PEG may be reacted with glutaric anhydridefollowed by reaction with 4-hydroxy-3-nitrobenzene sulfonic acid (HNSA)in the presence of dicyclohexyl carbodiimide to produce the activatedpolymer. HNSA is described by Bhatnagar et al., Peptides:Synthesis-Structure-Function, Proceedings of the Seventh AmericanPeptide Symposium, Rich et al. (eds.) (Pierce Chemical Co., RockfordIll., 1981), p. 97-100, and in Nitecki et al., High-Technology Route toVirus Vaccines (American Society for Microbiology: 1986) entitled “NovelAgent for Coupling Synthetic Peptides to Carriers and Its Applications.”

Specific methods of producing GH conjugated to PEG include the methodsdescribed in U.S. Pat. No. 4,179,337 on PEG-GH and U.S. Pat. No.4,935,465, which discloses PEG reversibly but covalently linked to GH.Other specific methods for producing PEG-GH include the following:

PEGylation with methoxypolyethylene glycol aldehyde (Me-PEG aldehyde) byreductive alkylation and purification is accomplished by adding to 2mg/mL of GH in PBS pH 7.0, 5 mM of Me-PEG aldehyde-5000 (molecularweight 5000 daltons) and 20 mM of NaCNBH3 and gently mixing at roomtemperature for 3 hours. Ethanolamine is then added to 50 mM toreductively amidate the remaining unreacted Me-PEG. The mixture isseparated on an anion-exchange column, FPLC Mono Q. The surplusunreacted Me-PEG does not bind to the column and can then be separatedfrom the mixture. Two main PEGylated GH fractions are obtained withapparent molecular weights of 30K and 40K on reduced SDS-PAGE, vs. 20Kof the unreacted GH. GH-GHBP complex is PEGylated in the same manner togive a derivative of 150K by gel filtration.

PEGylation with N-hydroxysuccinimidyl PEG (NHS-PEG) and purification areaccomplished by adding NHS-PEG at a 5-fold molar excess of the totallysine concentration of GH to a solution containing 2 mg/mL of GH in 50mM of sodium borate buffer at pH 8.5 or RBS at pH 7, and mixing at roomtemperature for one hour. Products are separated on a Superose 12 sizingcolumn and/or Mono Q of FPLC. The PEGylated GH varies in size dependingon the pH of the reaction from approximately 300 K for the reaction runat pH 8.5 to 40 K for pH 7.0 as measured by gel filtration. The GH-GHBPcomplex is also PEGylated the same way with a resulting molecular weightof 400 to 600 Kd from gel filtration.

PEGylation of the cysteine mutants of GH with PEG-maleimide isaccomplished by preparing a single cysteine mutant of GH bysite-directed mutagenesis, secreting it from an E. coli 16C9 strain(W3110 delta tonA phoA delta E15 delta (argF-lac) 169 deoC2 that doesnot produce the deoC protein and is described in U.S. Ser. No.07/224,520 filed 26 Jul. 1988, now abandoned, the disclosure of which isincorporated herein by reference) and purifying it on an anion-exchangecolumn. PEG-maleimide is made by reacting monomethoxyPEG amine withsulfo-MBs in 0.1 M sodium phosphate pH 7.5 for one hour at roomtemperature and buffer exchanged to phosphate buffer pH 6.2. Next GHwith a free extra cysteine is mixed in for one hour and the finalmixture is separated on a Mono Q column as in Me-PEG aldehyde PEGylatedGH.

As ester bonds are chemically and physiologically labile, it may bepreferable to use a PEG reagent in the conjugating reaction that doesnot contain ester functionality. For example, a carbamate linkage can bemade by reacting PEG-monomethyl ether with phosgene to give thePEG-chloroformate. This reagent could then be used in the same manner asthe NHS ester to functionalize lysine side-chain amines. In anotherexample, a urea linkage is made by reacting an amino-PEG-monomethylether with phosgene. This would produce a PEG-isocyanate that will reactwith lysine amines.

A preferred manner of making PEG-GH, which does not contain a cleavableester in the PEG reagent, is described as follows: Methoxypoly(ethyleneglycol) is converted to a carboxylic acid by titration with sodiumnaphthalene to generate the alkoxide, followed by treatment withbromoethyl acetate to form the ethyl ester, followed by hydrolysis tothe corresponding carboxylic acid by treatment with sodium hydroxide andwater, as reported by Buckmann et al., Macromol. Chem., 182: 1379-1384(1981). The resultant carboxylic acid is then converted to aPEG-N-hydroxysuccinimidyl ester suitable for acylation of GH by reactionof the resultant carboxylic acid with dicyclohexylcarbodiimide and NHSin ethyl acetate.

The resultant NHS-PEG reagent is then reacted with 12 mg/mL of GH usinga 30-fold molar excess over GH in a sodium borate buffer, pH 8.5, atroom temperature for one hour and applied to a Q Sepharose column inTris buffer and eluted with a salt gradient. Then it is applied to asecond column (phenyl Toyopearl) equilibrated in 0.3 M sodium citratebuffer, pH 7.8. The PEGylated GH is then eluted with a reverse saltgradient, pooled, and buffer-exchanged using a G25 desalting column intoa mannitol, glycine, and sodium phosphate buffer at pH 7.4 to obtain asuitable formulated PEG7-GH.

The PEGylated GH molecules and GH-GHBP complex can be characterized bySDS-PAGE, gel filtration, NMR, tryptic mapping, liquidchromatography-mass spectrophotometry, and in vitro biological assay.The extent of PEGylation is suitably first shown by SDS-PAGE and gelfiltration and then analyzed by NMR, which has a specific resonance peakfor the methylene hydrogens of PEG. The number of PEG groups on eachmolecule can be calculated from the NMR spectrum or mass spectrometry.Polyacrylamide gel electrophoresis in 10% SDS is appropriately run in 10mM Tris-HCl pH 8.0, 100 mM NaCl as elution buffer. To demonstrate whichresidue is PEGylated, tryptic mapping can be performed. Thus, PEGylatedGH is digested with trypsin at the protein/enzyme ratio of 100 to 1 inmg basis at 37° C. for 4 hours in 100 mM sodium acetate, 10 mM Tris-HCl,1 mM calcium chloride, pH 8.3, and acidified to pH<4 to stop digestionbefore separating on HPLC Nucleosil C-18 (4.6 mm×150 mm, 5μ, 100A). Thechromatogram is compared to that of non-PEGylated starting material.Each peak can then be analyzed by mass spectrometry to verify the sizeof the fragment in the peak. The fragment(s) that carried PEG groups areusually not retained on the HPLC column after injection and disappearfrom the chromatograph. Such disappearance from the chromatograph is anindication of PEGylation on that particular fragment that should containat least one lysine residue. PEGylated GH may then be assayed for itsability to bind to the GHBP by conventional methods.

The various PEGylation methods used produced various kinds of PEGylatedwild-type GH, with apparent molecular weights of 35K, 51K, 250K, and300K by size exclusion chromatography, which should be close to theirnative hydrodynamic volume. These were designated PEG 1-GH, PEG2-GH,PEG3-GH, and PEG7-GH, respectively. From the results of the trypticmapping, the PEG 1-GH and PEG2-GH both had the N-terminal 9-amino-acidfragment missing from the chromatogram and possibly PEGylated, whichcould be confirmed by the mass spectrometry of the big molecular speciesfound in the flow-through of the liquid chromatograph. From themolecular weight on SDS-PAGE, PEG 1-GH may have one PEG on theN-terminal amine, and the PEG2-GH may have two PEG molecules on theN-terminal amine, forming a tertiary amide. The PEG3-GH has about 5 PEGgroups per molecule based upon the NMR result, and on the tryptic map,at least five peptide fragments were missing, suggesting that they arePEGylated. The PEG7-GH molecule is believed to have 6-7 PEG groups permolecule based on mass spectrometry.

The sites for adding PEG groups to GH, and those that are preferredresidues for such conjugation, are N-terminal methionine orphenylalanine, lysine 38, lysine 41, lysine 70, lysine 140, lysine 145,lysine 158, and lysine 168. Two lysines that appeared not to bePEGylated were lysine 115 and lysine 172.

The GH to be used for in vivo administration must be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. The GHordinarily will be stored in lyophilized form or in solution.

Therapeutic GH compositions generally are placed into a container havinga sterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of GH administration is in accord with known methods. Examplesof parenteral administration include subcutaneous, intramuscular,intravenous, intraarterial, and intraperitoneal administration, or bysustained release systems as noted below. Subcutaneous and intravenousinjection or infusion is preferred.

Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers 22: 547-556 [1983]), poly(2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981] and Langer,Chem. Tech. 12: 98-105 [1982]), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release GH compositions also include liposomally entrapped GH.Liposomes containing GH are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamelar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal GH therapy.

An “effective amount” of GH to be employed therapeutically will depend,for example, upon the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. Typically, the clinician will administerthe GH until a dosage is reached that achieves the desired effect. Theprogress of this therapy is easily monitored by conventional assays.

In the treatment of congestive heart failure by GH, the GH compositionwill be formulated, dosed, and administered in a fashion consistent withgood medical practice. Factors for consideration in this context includethe particular mammal being treated, the clinical condition of theindividual patient, the site of delivery of the GH, the particular typeof GH, the method of administration, the scheduling of administration,and other factors known to medical practitioners. The “therapeuticallyeffective amount” of GH to be administered will be governed by suchconsiderations, and is the minimum amount necessary to ameliorate, ortreat the congestive heart failure, so as to increase ventricularcontractility and decrease peripheral vascular resistance, or toameliorate other conditions of similar importance in congestive heartfailure patients. Such amount is preferably below the amount that istoxic to the host or renders the host significantly more susceptible toinfections.

As a general proposition, the total pharmaceutically effective amount ofGH administered parenterally per dose will be in the range of about 1μg/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to a great deal of therapeutic discretion.More preferably, this dose is at least 0.01 mg/kg/day, and mostpreferably for humans between about 0.01 and 1 mg/kg/day. If givencontinuously, the GH is typically administered at a dose rate of about 1μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. Preferably, in humanpatients, a pharmaceutically effective amount of the GH administeredparenterally per dose will be in the range of about 10 to 100 microgramsper kilogram of patient body weight per day.

It is noted that practitioners devising doses of both IGF-1 and GHshould take into account the known side effects of treatment with thishormones.

As noted above, however, these suggested amounts of GH are subject to agreat deal of therapeutic discretion. The key factor in selecting anappropriate dose and scheduling is the result obtained, as indicatedabove.

EXAMPLES Use of GH to Treat Congestive Heart Failure Introduction

The goal of this study was to examine the cardiac effects of human GHtreatment in an animal model of congestive heart failure.

Methods

Male Sprague-Dawley (SD) rats (Charles River Breeding Laboratories,Inc., eight weeks of age) were acclimated to the facility for at least 1week before surgery. Rats were fed a pelleted rat chow and water adlibitum and housed in a light and temperature controlled room.

Coronary Arterial Ligation

Myocardial infarction was produced by left coronary arterial ligation asdescribed previously. Greenen, D. L. er al., J. Appl. Physiol. 63: 92-96(1987); Buttrick, P. et al., Am. J. Physiol. 260: 11473-11479 (1991).The rats were anesthetized with sodium pentobarbital (60) mg/kg, ip),intubated via tracheotomy, and ventilated by a respirator (HarvardApparatus Model 683). After a left-sided thoracotomy, the left coronaryartery was ligated approximately 2 mm from its origin with a 7-0 silksuture. Sham animals underwent the same procedure except that the suturewas passed under the coronary artery and then removed. All rats werehandled according to the “Position of the American Heart Association onResearch Animal Use” adopted Nov. 11, 1984 by the American HeartAssociation. 4-6 weeks after ligation myocardial infarction coulddevelop heart failure in rats. In clinical patients, myocardialinfarction or coronary artery disease is the most common cause of heartfailure. Congestive heart failure in this model reasonably mimicscongestive heart failure in most human patients.

Electrocardiograms

One week after surgery, electrocardiograms were obtained under lightmetofane anesthesia to document the development of infarcts. The ligatedrats of this study were subgrouped according to the depth andpersistence of pathological Q waves across the precordial leadsButtrick, P. et al., Am. J. Physiol. 260: 11473-11479 (1991); Kloner, R.A. et al., Am Heart J. 51: 1009-1013 (1983). This provided a grossestimate of infarct size and assured that large and small infarcts werenot differently distributed in the ligated rats treated with GH andvehicle. Confirmation was made by precise infarct size measurement.

GH Administration

Four weeks after surgery, recombinant human GH (1 mg/kg twice a day for15 days) (Genentech, Inc., South San Francisco, Calif.) or salinevehicle was injected subcutaneously in both ligated rats and shamcontrols. Previous studies have shown that this dose of human GH canproduce significant anabolic effects in rats. Moore et al.,Endocrinology 122: 2920-2926 (1988); R. Clark and M. Cronin, U.S. Pat.No. 5,126,324 (1992). Body weight (BW) was measured twice a week duringthe treatment. See FIG. 1. GH can be administered in saline or water asvehicles.

Catheterization

After 13 day treatment of GH or vehicle, rats were anesthetized withpentobarbital sodium (50 mg/kg, intraperitoneal). A catheter (PE-10fused with PE 50) filled with heparin-saline solution (50/U/ml) wasimplanted into the abdominal aorta through the right-femoral artery formeasurement of arterial pressure and heart rate. A second catheter (PE50) was implanted into the right atrium through the right jugular veinfor measurement of right atrial pressure and for saline injection. Formeasurement of left ventricular pressures and contractility (dP/dt), athird catheter (PE 50) was implanted into the left ventricle through theright carotid artery. For the measurement of cardiac output by athermodilution method, a thermistor catheter (Lyons Medical InstrumentCo., Sylmar, Calif.) was inserted into the aortic root. The catheterswere exteriorized at the back of the neck with the aid of a stainlesssteel wire tunneled subcutaneously and then fixed. Following catheterimplantation, all rats were housed individually.

Hemodynamic Measurements

One day after catherization, the thermistor catheter was processed in amicrocomputer system (Lyons Medical Instrument Co.) for cardiac outputdetermination, and the other three catheters were connected to a ModelCP-10 pressure transducer (Century Technology Company, Inglewood,Calif., USA) coupled to a Grass Model 7 polygraph (Grass Instruments,Quincy, Mass., USA). Mean arterial pressure (MAP), systolic arterialpressure (SAP), heart rate (HR), right atrial pressure (RAP), leftventricular systolic pressure (LVSP), left ventricular mean pressure(LVMP), left ventricular end-diastolic pressure (LVEDP) and leftventricular maximum (dP/dt) were measured in conscious, unrestrainedrats. For measurement of cardiac output, 0.1 ml of isotonic saline atroom temperature was injected as a bolus via the jugular vein catheter.The thermodilution curve was monitored by VR-16 simultrace recorders(Honeywell Co., NY) and cardiac output (CO) was digitally obtained bythe microcomputer. Stroke volume (SV)=CO/HR; Cardiac index (CI)=CO/BW;Systemic vascular resistance (SVR)=MAP/CI.

After measurement of these hemodynamic parameters, 1 ml blood wascollected through the arterial catheter. Serum was separated and storedat −70° C. for measurement of GH and IGF-1.

At the conclusion of the experiments, rats were anesthetized withpentobarbital sodium (60 mg/kg) and the heart was arrested in diastolewith intra-atrial injection of KCl (1 M). The heart was removed, and theatria and great vessels were trimmed from the ventricle. The ventriclewas weighed and fixed in 10% buffered formalin. See FIG. 1, Bottom.

All experimental procedures were approved by Genentech's InstitutionalAnimal Care and Use Committee before initiation of the study.

Infarct Size Measurements

The right ventricular free wall was dissected from the left ventricle.The left ventricle was cut in four transverse slices from apex to base.Five micrometer sections were cut and stained with Massons' trichromestain and mounted. The endocardial and epicardial circumferences of theinfarcted and non-infarcted left ventrical were determined with aplanimeter Digital Image Analyzer. The infarcted circumference and theleft ventricular circumference of all four slices were summed separatelyfor each of the epicardial and endocardial surfaces and the sums wereexpressed as a ratio of infarcted circumference to left ventricularcircumference for each surface. These two ratios were then averaged andexpressed as a percentage for infarct size.

Hormone Assays

Serum human GH was measured by a sensitive ELISA. A. Celniker (abstr)Am. Endocrin Soc., A. Celniker et al., Endocrinol. Metab. 68(2): 469(1989); Greenen, D. L. et al., J. Appl. Physiol. 63: 92-96 (1987). Thisassay does not detect rat GH. Total serum IGF-1 was measured afteracid-ethanol extraction by radioimmunoassay, for example, RIA describedby Furlanetto et al., J. Clin. Invest 60: 648-657 (1977); Bala andBhaumick, J. Clin. Endocrinol. and Metab. 49: 770-777 (1979); Zapf etal., J. Clin. Invest. 68: 1321-1330 (1981); Hall et al., J. Clin. Endo.Metab. 48: 271-278 (1979); EP 292,656., using human IGF-1 (GenentechM3-RD1) as the standard and a rabbit anti-IGF-1 polyclonal antiserum.The acceptable range was 1.25-40 ng/ml, while the intra and inter-assayvariability were 5-9% and 6-15%, respectively. See FIG. 2.

Statistical Analysis

Results are expressed as mean±SEM. Two way and one way analysis ofvariance was performed to assess differences in parameters betweengroups. Significant differences were then subjected to post hoc analysisusing the Newman-Keuls method. P<0.05 was considered significant.

Results

The mean BW before treatment of GH or vehicle was not different betweenthe experimental groups (FIG. 1A). There was significantly greaterincrease in BW following GH treatment for both sham and ligated rats(FIG. 1B). LCA ligation caused a significant increase in the ratio ofventricular weight (VW) to BW, while GH treatment did not alter thisratio significantly (FIG. 1C).

GH treatment significantly increased serum levels of human GH and IGF-1in both sham ligated rats (FIGS. 2A and 2B). The GH-induced increment inserum levels of human GH and IGF-1 was not significantly differentbetween sham and ligated rats.

Infarct size in ligated rats was not different between thevehicle-treated group (33.2±2.2% of the left ventricle) and theGH-treated group (31.4±2.6% of the left ventricle).

LCA ligation resulted in significant decreases in MAP in thevehicle-treated rats but not in the GH-treated rats (FIG. 3A). GHtreatment significantly decreased MAP in the sham rats but not in theligated rats. LCA ligation was associated with significant reductions inSAP in both vehicle-treated and GH-treated rats (FIG. 3B). However, theligation-induced decrease in SAP was significantly greater in thevehicle-treated rats than that in the GH-treated rats. GH administrationdid not alter SAP significantly in the sham rats. Neither LCA ligationnor GH treatment altered HR significantly (FIG. 3C).

LCA ligation significantly lowered left ventricular dP/dt and LVSP inthe vehicle-treated rats but not in the GH-treated rats (FIGS. 4A andB).

GH treatment increased dP/dt and LVSP in the ligated rats but not in thesham rats.

In the vehicle-treated animals, LVEDP was significantly elevated in theligated group compared to sham controls (FIG. 4C). In the GH-treatedanimals, however, there was no significant difference in LVEDP betweenthe ligated and sham group. GH administration decreased LVEDPsignificantly in the ligated rats but not in the sham rats.

LCA ligation produced significant reductions in CI and SVI in thevehicle treated rats but not in the GH-treated rats (FIGS. 5A and B). GHadministration significantly increased CI and SVI in the ligated ratsbut not in sham controls. There was no significant difference in SVRbetween the ligated and sham rats, while GH treatment significantlylowered SVR in the ligated rats and tended to lower SVR in sham controls(FIG. 5C).

In the current study, 6 weeks after left coronary artery (LCA) ligation,rats treated with vehicle exhibited significant decreases in LVSP,dP/dt, CI, and SVI and increases in LVEDP compared to the sham controls.These results indicate that congestive heart failure occurred in thisanimal model of myocardial infarction primarily due to a decrease inventricular contractility. GH treatment at the dose of 2 mg/kg/day for15 days significantly increased LVSP, dP/dt, CO, and SVI and reducedLVEDP and SVR in the LCA ligated rats. This result demonstrates thatadministration of GH improves cardiac function by increasing ventricularcontractility and decreasing peripheral vascular resistance incongestive heart failure. In sham rats, however, GH administration atthis dose did not significantly alter cardiac function except slightlylowering arterial pressure and peripheral vascular resistance.

It would be reasonably expected that the rat data herein may beextrapolated to horses, cows, humans and other mammals, correcting forthe body weight of the mammal in accordance with recognized veterinaryand clinical procedures. Using standard protocols and procedures, theveterinarian or clinician will be able to adjust the doses, scheduling,and mode of administration of GH and its variants to achieve maximaleffects in the desired mammal being treated. Humans are believed torespond in this manner as well.

1. A method of treating a mammal exhibiting congestive heart failurecomprising administering to said mammal an effective amount of growthhormone.
 2. The method of claim 1 wherein said growth hormone is humangrowth hormone.
 3. The method of claim 1 wherein said mammal is human.4. The method of claim 3 wherein said effective amount is in the rangeof 10-100 micrograms per kilogram of body weight per day.
 5. The methodof claim 3 wherein said administering is subcutaneous or intravenous. 6.The method of claim 1 wherein said congestive heart failure results frommyocardial infarction.